The overall objective of this U01 application is to develop an orally active, optimized lead compound for the treatment of amnestic mild cognitive impairment (aMCI). The proposed therapy is based on the observation that memory loss in aMCI, a borderline condition between normal aging and Alzheimer's Disease (AD), is associated with excess activity in the CA3/dentate gyrus (DG) region of the hippocampus. Reducing excess activity, or normalizing it, is expected to improve memory in these patients. Preclinical studies in an animal model of this condition, in which hippocampal CAS neurons are hyperactive in aged rats with memory loss, demonstrates that selective GABAA a5 receptor agonists are effective therapeutic agents to improve memory. We have identified several different chemical series that are selective for GABAA a5 receptors. Compounds within these series were originally developed by large pharmaceutical companies to optimize inverse agonist activity with the objective of improving cognition. This approach was not efficacious in the clinic; indeed the science supporting our proposed work would predict that such an approach would fail. Still these chemical series have drug like properties that provide a starting point for optimization of selective a5 receptor agonists. Under the specific aims we will use established in vitro assays in a medicinal chemistry program to optimize selectivity and agonist efficacy for GABAA a5 subunit containing receptors and conduct early ADME and toxicology work to determine suitability for administration to animals. In vivo studies will then be performed in an animal model of memory loss in aging that mirrors many features observed in aged humans, particularly aMCI. Companion studies to determine in vivo receptor occupancy using multiple tracers with liquid chromatography coupled to tandem mass spectral detection (LC/MS/MS) will also be conducted to validate engagement of target GABAA a5 receptors, as well as selectivity for that receptor subtype, at doses that are behaviorally efficacious. In the final phase of the project we will complete all materials, including pharmacokinetics and toxicology, good manufacturing practice (GMP) synthesis and formulation for lead GABAA a5 receptor agonist filing with the FDA. RELEVANCE (See instructions):